a Department of Internal Medicine, Sint Franciscus Gasthuis, Rotterdam, The Netherlandsb Tytgat Institute for Liver and Intestinal Research, Amsterdam Medical Centre, Amsterdam, The Netherlandsc Unitat de Recerca en Lípids i Arteriosclerosi, CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Institutd’Investigació Sanitària Pere Virgili, Universitat Rovira i Virgili, Reus, Spaind U-705 CIBERER, Barcelona, Spain
Received 24 September 2013; accepted 18 November 2013Available online 27 December 2013
AbstractBackground: The microsomal triglyceride transfer protein (MTP) is involved in hepatic andintestinal apoB secretion. We studied the effect of the functional MTP−493G/T polymorphismon fasting and postprandial lipoproteins in patients with familial combined hyperlipidemia (FCH)before and after treatment with atorvastatin.Methods: Eight FCH heterozygote carriers of the rare −493T allele were compared to 9 matchedFCH homozygotes for the wild-type allele in a pilot study. Oral fat loading tests were carried outto measure triglycerides (TG) and apo B48 and B100 in the different fractions of triglyceride-richlipoproteins (TRLs) before and after treatment with atorvastatin.Results: Before treatment, TG were similar between the −493T allele carriers and non-carriers.In the T-allele carriers, a trend was observed for increased postprandial apo B48 and B100concentrations in Sf >400 and Sf 60---400 compared to non-carriers. After treatment, fasting andpostprandial TG were significantly lowered in carriers of the T allele, but atorvastatin had noeffect on postprandial TG in non-carriers. Atorvastatin resulted in similar reductions of apo B48
and B100 in TRLs in both groups.Conclusion: The MTP-493G/T polymorphism modulates postprandial apo B48 and apo B100 of
ecreases postprandial TG in T-allele carriers with FCH.
amilial combined hyperlipidemia (FCH) is a complex, poly-enic disease characterized by increased VLDL productionnd decreased remnant clearance.1---6 Multiple genes haveeen associated with FCH, but none of them has beenully informative.2,7---11 The microsomal triglyceride transferrotein (MTP) has a key function in the intracellular lipi-ation and secretion of apolipoprotein (apo) B by the livernd intestine.12---14 MTP shuttles triglycerides (TG) from themooth endoplasmic reticulum to the rough endoplasmiceticulum, where apo B is synthesized and mature TG-richipoproteins are produced. Mutations in the MTP gene leado MTP deficiency associated with abetalipoproteinemia inomozygotes.12---14
Besides structural mutations leading to abetalipopro-einemia, the MTP gene seems polymorphic with multipleubtle changes in the MTP gene structure. The func-ional MTP−493G/T polymorphism has been associatedith increased transcriptional activity with the T vari-nt affecting TG metabolism.15,16 Increased postprandialeneration of smaller, intestinally produced lipoproteinsnd reduced fasting TG plasma concentrations have beeneported with the rare MTP−493T allele, but results wereot always consistent.17---20 Furthermore, the MTP−493Tolymorphism has been associated with decreased TGevels in untreated patients with heterozygous famil-al hypercholesterolemia.16,21 In addition, the MTP−493T
olymorphism increased the TG lowering effect of ator-astatin in male subjects with heterozygous familialypercholesterolemia.21 Moreover, homozygous carriers ofhe T-allele showed a stronger reduction of fasting TG after
arwe
diet low in saturated fatty acids and high in monounsatu-ated fatty acids and polyunsaturated fatty acids for a periodf 3 months.22 The T allele has also been associated withncreased insulin resistance and higher TG concentrationsn males with the metabolic syndrome.23 Insulin resistancend elevated TG are characteristics of FCH and the MTP geneas been proposed as a candidate gene for the developmentf FCH. However, this could not be confirmed in a cohortf patients with the FCH phenotype based on fasting lipidrofiles.24
This pilot study was initiated to evaluate whether theTP−493G/T polymorphism is associated to postprandialG, apo B48 and apo B100 changes in patients with FCHnd whether the MTP−493G/T polymorphism influenceshe effects of atorvastatin on postprandial lipemia in FCHatients.
aterial and methods
ubjects
he Independent Ethics Committee of the University Medi-al Center Utrecht approved the study protocol andritten informed consent was obtained from each partic-
pant. Seventeen unrelated FCH patients were recruitedy genotype: participants were either heterozygous forhe MTP−493T allele or homozygous for the MTP−493G
llele. FCH was diagnosed with the following crite-ia: subjects were known with primary hyperlipidemiaith varying phenotypic expression and they had alllevated plasma apoB concentrations (>1.20 g/l) when
lial c
t(Sifiawnottbasaicww(2wwTasQbpmomiil
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AmtsentFwb1aPC(
R
MTP gene polymorphisms and postprandial lipidemia in fami
untreated; at least one first degree relative had a dif-ferent hyperlipidemic phenotype, and each index subjecthad a positive family history of premature cardiovas-cular disease disease defined as myocardial infarctionor cerebrovascular disease before the age of 60. Inaddition, the patients fulfilled the following inclusioncriteria: absence of xanthomas, and secondary factors asso-ciated to hyperlipidemia, body mass index (BMI) <30 kg/m2,absence of apo E2/E2 genotype and no use of more than 3units of alcohol per day.
Study design and treatment protocol
At inclusion, six out of seventeen FCH patients were usinglipid-lowering drugs (atorvastatin 10 mg, 40 mg, 80 mg, orsimvastatin 20 mg once daily). They stopped their med-ication 4 weeks before the first oral fat loading testwas performed. After the first oral fat loading testthe FCH patients were treated for 16 weeks with ator-vastatin. Atorvastatin was chosen since statins are thefirst choice of drug treatment in FCH. Statins increasethe fractional catabolic rate of both LDL and VLDLtogether with a slight reduction of hepatic VLDL secre-tion. The initial dose of atorvastatin was 10 mg oncedaily. Every four weeks, the patients visited the outpatientclinic and fasting plasma lipids and apolipoproteins weremeasured. When plasma TG concentrations were above2.00 mmol/l and/or total cholesterol levels were above6.5 mmol/l, the atorvastatin dose was doubled, up to amaximum dose of 80 mg after 12 weeks. After 16 weeksof atorvastatin, a second oral fat loading test was per-formed.
Oral fat loading test
Cream was used as a fat source; this is a 40% (w/v) fatemulsion with a P/S ratio of 0.06, which contains 0.001%(w/v) cholesterol, 2.8% (w/v) carbohydrates and 60 g/l dex-trose. After an overnight fasting period of 10 hours, thesubjects ingested cream (50 g/m2) and were allowed to drinkonly water and sugar-free tea during the following 8 hours.Peripheral blood samples were obtained in sodium EDTA(2 mg/ml) before (t = 0 h), and at 1-hourly intervals up to8 h.
Analytical methods and laboratory techniques
Blood was placed on ice and centrifuged immediately for15 min at 800 g at 4 ◦C. After centrifugation, a proteaseinhibitor was added to the plasma as described.25 Plasmasamples were stored at −20 ◦C immediately after centrifu-gation. Apo E genotypes were determined as describedearlier.26 MTP genotypes were determined as describedby Karpe and co-workers.15,18 TG and total cholesterolwere measured in duplicate by a commercial colorimetricassay (GPO-PAP and CHOD-PAP, Roche, respectively). HDL-
cholesterol was determined as described earlier by Bursteinusing the phosphotungstate/MgCl2 method.27 Lipoproteinsat time-points t = 0, 2, 4, 6 and 8 h were subfractionated byultracentrifugation as described in detail.25,28
G
Ap
ombined hyperlipide 51
ApoB48 and apoB100 in Svedberg flotation (Sf) frac-ions representative for chylomicrons (Sf > 400), VLDL1Sf 400---60) and VLDL2 (Sf 60---20) were quantitated byDS-PAGE.25,28,29 Briefly, samples of each fraction were delip-dated in a methanol/diethylether solvent system. After therst step, debris was removed with ice cold diethylether,nd the proteins were dried by evaporation. The materialas dissolved in sample buffer. Aliquots for apo B determi-ation were stored at −80 ◦C, and assayed within 3 monthsn 3---5% SDS-PAGE. The amount of apoB100 in the TRL frac-ions is usually too high to quantitate directly by SDS-PAGE;herefore, each sample was diluted 20 times with sampleuffer and then loaded on the gel. For quantification ofpo B48, each sample was loaded on the gel undiluted. Thetandard curve was made by delipidated LDL with knownbsolute amounts of proteins. In order to assess the equal-ty of chromogenicities of apo B48 and apo B100, humanhylous ascites, containing significant amounts of apoB48,as also delipidated and run on each gel.25 The proteinsere stained with the Colloidal Blue Staining kit from Novex
Invitrogen, Carlsbad, CA, USA), containing Coommassie G-50, and destained by washing the gels at least four timesith distilled water. After geometrical calibration, the gelsere scanned with a B/W CCD camera type XC-77CE (Sony,
okyo, Japan). For quantification of apo B48 and apo B100PC-based image analysis system was used (KS400 ver-
ion 3.0 software, Carl Zeiss Vision, Oberkochen, Germany).uantification of apo B48 and apo B100 was performedy a technician who was blinded to the code of the sam-les. The reproducibility of the method, calculated as 100%inus the coefficient of variation is 92.7%. The recovery
f the apoB samples ranged from 70 to 80%. ApoAI waseasured by nephelometry using apoAI polyclonal antibod-
es (OUED 14/15, Behring Diagnostics NV). The HOMA-IRndex (=glucose (mmol/l) × insulin (mU/l)/22.5) was calcu-ated.
tatistical methods
ll values in the text and tables are expressed asean ± standard deviation (SD). Mean ± standard error of
he mean (SEM) are used in the figures. Due to the smallample size, the groups were divided based on the pres-nce of the T allele. Mean differences between carriers andon-carriers were calculated by the independent-samples-test. Mean differences between untreated and treatedCH subjects were calculated by paired-samples t-test. TGere logarithmically transformed to obtain a normal distri-ution. Statistical calculations were performed using PASW8.0 (IBM SPSS Inc. Chicago, IL, USA). Calculations for therea under the curves (AUC) were performed with Graph-ad Prism version 5.0 (GraphPad Software Inc. San Diego,A, USA). Statistical significance was reached when P < 0.05two-tailed).
esults
eneral characteristics
total of seventeen FCH patients were included. Nineatients were homozygote for the normal G allele, and
52 B. Klop et al.
Table 1 Baseline characteristics in familial combined hyperlipidemia patients in non-carriers and MTP−493T-allele carriersbefore treatment with atorvastatin. Data are given as mean ± standard deviation unless stated otherwise.
patients were heterozygote carriers of the MTP−493Tllele. Baseline characteristics were similar between non-arriers and the T allele carriers: age (45.6 ± 10.6 yearss 43.4 ± 11.4 years), male gender (55.6% vs 37.5%),ody mass index (25.6 ± 2.2 kg/m2 vs 25.8 ± 3.3 kg/m2)
nd HOMA-IR (3.07 ± 1.87 vs 2.65 ± 0.92). None of theatients expressed the apo E2/E2 genotype and theistribution of the apo E genotype was comparableetween non-carriers and the T allele carriers. There
M
Tp
7
7 8
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sma
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mol
/l)
TG
in S
F >
400
(m
mol
/l)
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in S
f 60-
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(mm
ol/l)
TG
in S
f 20-
60 (
mm
ol/l)
Time (hours)
Time (hours)
493T-aNon-ca
A B
C D
igure 1 Mean postprandial changes of plasma triglycerides (TG) (nd 20---60 (D) for untreated FCH patients in MTP−493T-allele carrieoad. No significant differences were found in plasma TG or any of th
ere no significant differences in the variables shown inable 1.
ostprandial lipid metabolism according to the
TP−493G/T polymorphism
otal cholesterol remained unchanged in both groups post-randially. Postprandial triglyceridemia showed a similar
86420
1
0,8
0,6
0,4
0,2
00 2 4 6 8
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2
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1
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Time (hours)
Time (hours)
llele carriersrriers
A), TG in Svedberg flotation fractions (Sf) >400 (B), 60---400 (C)rs (open dots) and non-carriers (closed dots) during an oral fate subfractions between −493T-allele carriers and non-carriers.
MTP gene polymorphisms and postprandial lipidemia in familial combined hyperlipide 53
2 20
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Time (hours)
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apoB
48 in
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(m
g/l)
apoB
48 in
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f > 4
00 (
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l)ap
oB10
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0-40
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g/l)
apoB
100
in S
f 20-
60 (
mg/
l)
apoB
48 in
Sf 2
0-60
(m
g/l)
Apolipoprotein B48 Apolipoprotein B100
493T-allele carriersNon-carriers
A D
B
C
E
F
Figure 2 Mean postprandial changes of apolipoprotein (apo) B48 and B100 in the Svedberg flotation fractions (Sf) >400 (A and D),60---400 (B and E) and 20---60 (C and F) fraction in untreated FCH patients in MTP−493T-allele carriers (open dots) and non-carriers(closed dots) during an oral fat load. No significant differences were found in apoB48 or apoB100 in any of the subfractions between
lcPi3seiwaaarr((
−493T-allele carriers and non-carriers.
pattern for the T allele carriers and non-carriers (Fig. 1A). Inaddition, no significant differences were found between theT allele carriers compared to the non-carriers in postpran-dial TG concentrations in the different TG-rich lipoproteinfractions (Fig. 1B---D). A non-significant trend for increasedpostprandial apo B48 and B100 concentrations in Sf >400and Sf 60---400 in FCH carriers of the MTP−493T allele wasobserved (Fig. 2).
Treatment effects of atorvastatin
After 16 weeks, at the end of the titration period, 2 patientsused 10 mg, 5 patients used 20 mg, 1 patient used 40 mg and9 patients used 80 mg of atorvastatin. In the fasted statetreatment with atorvastatin lowered plasma total choles-terol, LDL-C and apo B in the T allele carrier FCH patientsas well as in the non-carriers (Table 2). Additionally, fas-
ting plasma TG were significantly lowered by atorvastatinin the T-allele carriers, whereas HDL-C was increased bythe non-carrier FCH patients after treatment with atorvas-tatin.
D
Pp
Treatment with atorvastatin resulted in a significantlyowered AUC for postprandial plasma TG in the T allelearriers (21.97 ± 8.52 mmol h/l vs 29.94 ± 13.51 mmol h/l,= 0.02), but postprandial triglyceridemia was unaltered
n the non-carrier FCH patients (32.07 ± 16.01 mmol h/l vs5.40 ± 12.87 mmol h/l, P = 0.49), despite an approximatelyimilar reduction in fasting TG (Fig. 3). The TG loweringffect of atorvastatin in non-carriers was most notablen the late postprandial phase (after 4---8 h). Treatmentith atorvastatin showed in both carriers of the T-allelend non-carriers a non-significant trend with loweredpo B48 in Sf >400 and lowered apo B100 in Sf >400nd Sf 60---400 (Figs. 4 and 5). Finally, the postprandialesponse for apo B100 in Sf 20---60 was significantlyeduced after treatment with atorvastatin in non-carriers221.7 ± 106.4 mg h/l vs 140.2 ± 62.9 mg h/l, P = 0.04)Fig. 5F).
iscussion
atients with FCH are characterized by exaggerated post-randial lipemia with a defective clearance of chylomicrons
54
7
7 8
6
6
5
5
4
4
3
3
2
2
Time (hours)
Non-carriers
T-allele carriers
Time (hours)
1
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6
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7 86543210
Pla
sma
TG
(m
mol
/l)P
lasm
a T
G (
mm
ol/l)
A
B
Before treatment
After treatment
Figure 3 Mean postprandial changes of plasma triglycerides(TG) before (closed dots) and after treatment (open dots) withatorvastatin in FCH patients in non-carriers (A) and MTP−493T-allele carriers (B) during an oral fat load. The total area underthe TG curve was significantly reduced after treatment inMTP−493T-allele carriers (P = 0.02) but not in non-carriers.
ailciiptutiBnpn
sdoiSeHbeSMa
orteeitpriaficw
Table 2 Changes in fasting lipid parameters before and after treapatients in non-carriers and carriers of the MTP−493T-allele. Data ar
* Significant change in corresponding value before versus after treatm** Significant change in corresponding value before versus after treatm
B. Klop et al.
nd their remnants. The major metabolic disturbancen FCH is a hepatic overproduction of hepatic TG-richipoproteins,30 but intestinal overproduction of chylomi-rons has not been excluded yet.1 In other disorders withnsulin resistance, for example type 2 diabetes mellitus,ntestinal overproduction has also been proposed.31---33 Theresent pilot study suggests increased postprandial concen-rations for apo B48 and B100 in the larger lipoproteins ofntreated T-carriers. These results were not surprising sincehe T allele is known for its increased transcriptional activ-ty of the MTP gene, which is associated with increased apo48 concentrations.15,18,22 Our results indicate an increasedumber of smaller apo B containing lipoproteins in FCHatients with the T-allele, although the differences wereot significant due to the small number of participants.
After treatment with atorvastatin, carriers of the T-allelehowed a significant reduction in both fasting and postpran-ial TG in contrast to non-carriers. In addition, carriersf the T-allele showed a trend with a greater reductionn large VLDL (Sf >400) after treatment with atorvastatin.tatin therapy has been shown to lower intestinal MTP mRNAxpression in diabetic and non-diabetic subjects.34 In vitroepG2 cells showed a reduction in MTP mRNA transcriptiony approximately 50% when they were cultured in the pres-nce of pravastatin to induce a sterol depleted condition.35
ince the T-allele is associated with higher transcriptionalTP activity,15 the statin inhibiting effect was probably morepparent in T-allele carriers.34
Our results show a more pronounced treatment responsen postprandial lipemia in T-allele carrying FCH patientseceiving atorvastatin. Two other studies have investigatedhe MTP−493G/T polymorphism in relation to treatmentffects of lipid lowering therapy.21,22 Patients with het-rozygous familial hypercholesterolemia showed a reductionn plasma TG after treatment with atorvastatin, buthe TG reduction was dependent on the MTP−493G/Tolymorphism.21 The T-allele was associated with a greatereduction in TG after treatment with atorvastatin, but onlyn males. In addition, a 3-month diet low on saturated fattycids and high on monounsaturated and polyunsaturated
atty acids lowered TG, but its effect was more pronouncedn carriers of the T-allele and especially in homozygousarriers of the T-allele.22 These results are in concordanceith our findings.
tment with atorvastatin in familial combined hyperlipidemiae given as mean ± standard deviation unless stated otherwise.
ent with atorvastatin (P < 0.05).ent with atorvastatin (P < 0.01).
MTP gene polymorphisms and postprandial lipidemia in familial combined hyperlipide 55
1,8
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apoB
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T-allele carriers
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1616
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Figure 4 Mean postprandial changes of apolipoprotein (apo) B48 and B100 in the Svedberg flotation fractions (Sf) >400 (A and D),60---400 (B and E) and 20---60 (C and F) fraction in untreated (closed dots) FCH carriers of the MTP−493T-allele and after treatmentwith atorvastatin (open dots) during an oral fat load. No significant changes were found in the AUC of the different fractions for apoB48 and B100 after treatment. However, treatment with atorvastatin showed a trend with lowered apo B48 in Sf >400 and loweredapo B100 in Sf >400 and Sf 60---400.
cligdAtrFr
Bw
MTP is required for the assembly of both intestinal andhepatic derived apo B containing lipoproteins. Despite simi-lar postprandial TG levels between the different genotypes,increased plasma apo B48 concentrations have been foundin the low Sf 20---60 fraction for homozygous carriers of theMTP−493T-allele in a previous study.18 We could not repro-duce these differences in apo B48 concentrations betweenT-allele carrying and non-carrying FCH patients. However,we observed a trend of increased postprandial concentra-tions of apo B48 and B100 in the larger lipoproteins incarriers of the T-allele. This discrepancy between previousresults and our study may be explained by the absence ofhomozygote carriers of the MTP−493T-allele in our study
population or due to the limited number of study partici-pants to reach conclusive results.
Several genes have been proposed to modulate theFCH phenotype,2,24 however MTP has not been found to
mapa
ontribute to the FCH phenotype in a previous study.24 Thisinkage study including 151 FCH patients out of 481 fam-ly members did not show an association between the MTPene and the FCH phenotype in the fasting state, but theyemonstrated a strong association between FCH and the apoI-CIII-AIV gene cluster and the lecithin:cholesterol acyl-ransferase (LCAT) locus.24 Our data do not question theesults of this study, but we did show subtle differences inCH patients with a different MTP−493G/T polymorphism inelation to atorvastatin treatment.
In conclusion, a trend of increased postprandial apo48 and B100 in FCH patients with the MTP−493T alleleas observed compared to non-carriers. After treat-
ent with atorvastatin FCH patients with the MTP−493T
llele showed an increased reduction in fasting and post-randial TG compared to non-carriers of the variantllele.
56 B. Klop et al.
Before treatmentAfter treatment
Time (hours) Time (hours)
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oB10
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100
in S
f 60-
400
(mg/
l)ap
oB10
0 in
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0 >
400
(m
g/l)
D
Non-carriers
E
F
Figure 5 Mean postprandial changes of apolipoprotein (apo) B48 and B100 in the Svedberg flotation fractions (Sf) >400 (A and D),60---400 (B and E) and 20---60 (C and F) fraction in untreated (closed dots) and after treatment with atorvastatin (open dots) in FCHn 20---( trend>
E
PdwEM
Cftst
Rosd
C
T
A
TgtG
R
on-carriers during an oral fat load. The AUC for apoB100 in SfP = 0.04). Additionally, treatment with atorvastatin showed a400 and Sf 60---400.
thical responsibilities
rotection of human and animal subjects. The authorseclare that the procedures followed were in accordanceith the regulations of the responsible Clinical Researchthics Committee and in accordance with those of the Worldedical Association and the Helsinki Declaration.
onfidentiality of Data. The authors declare that they haveollowed the protocols of their work centre on the publica-ion of patient data and that all the patients included in thetudy have received sufficient information and have givenheir informed consent in writing to participate in that study.
ight to privacy and informed consent. The authors havebtained the informed consent of the patients and /orubjects mentioned in the article. The author for correspon-ence is in possession of this document.
60 was significantly reduced after treatment with atorvastatinwith lowered apo B48 in Sf >400 and lowered apo B100 in Sf
onflict of interest
he authors declare that there are no conflicts of interest.
cknowledgements
his study was supported by an unrestricted educationalrant from Pfizer Inc and by the Research Foundation ofhe Department of Internal Medicine of the Sint Franciscusasthuis.
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